Diabetes is a devastating autoimmune disease of immense proportions. It is characterized by an impaired glucose metabolism that leads to, among other things an elevated blood glucose level (hyperglycemia) in diabetic patients. Diabetes is classified into type 1, or insulin dependent diabetes mellitus (IDDM), which arises when a patient's β-cells cease producing insulin in their pancreatic glands, and type 2, or non-insulin dependent diabetes mellitus (NIDDM), which occurs in patients with an impaired insulin metabolism and β-cell malfunction. NIDDM usually takes decades to develop and is characterized sequentially by hyperinsulinemia, elevated triglycerides, high blood glucose and finally in late stages β cell fatigue, where insulin levels drop precipitously usually requiring insulin administration to the patient. In IDDM patients, the β-cells are selectively destroyed by an autoimmune process that involves lymphocyte infiltration. Early in the course of NIDDM, β-cell mass increases to meet the demand for more insulin. Loss of β-cell mass may then occur as NIDDM advances.
Individuals at risk for developing IDDM can be identified by certain techniques. Those at risk for NIDDM are identifiable through family history and measurement of insulin resistance. However, little is known about the natural history of β-cell mass, turnover and cell lifetime, or the course of inflammation in diabetes. This is attributable to the highly heterogeneous nature of the pancreas, difficulties in its biopsy, and the low volume of β-cell mass (only 1-2% of the organ). Although insulin secretory capacity can be measured, it poorly reflects β-cell mass. There is therefore a substantial need for diagnostic methods that would enable (i) high-risk individuals to be monitored prior to onset of diabetes; (ii) diabetes patients to be monitored over the course of their disease to determine the exact stage of their disease; and (iii) also monitoring responses to therapy.
IDDM is being successfully treated using pancreas transplantation, and researchers are now able to achieve insulin independence in patients by transplanting healthy, functioning isolated pancreatic islets into patients. There is a great clinical need to identify the location, number, viability, growth and function of these grafts, and to non-invasively monitor their response to immune modulating therapy, using imaging. Another promising IDDM therapy is the transplantation of isolated, polymer encapsulated pancreatic islets. Primary islet dysfunction after islet transplantation is often encountered, but the causes are still poorly understood. Again, new diagnostic techniques that could assess local inflammation around engrafted islets would be of great clinical benefit.
Current therapeutics for Type 1 diabetics are insulin or insulin mimetics, while most type 2 diabetic patients are treated either with agents that stimulate β-cell function or enhance the patient's tissue sensitivity towards insulin. Several classes of drugs are available for diabetes therapy. These include: insulin, or insulin mimetics; insulin sensitizers including (a) biguanides such as Metformin (b) retinoid-X-receptor (RXR) and peroxisome proliferator activated receptor (PPAR) agonists, such as the Thiazolidinedione (glitazone) and PPAR-γ agonists, e.g., Rosiglitazone and Troglitazone; (c) sulfonylureas (SU), such as Gliclazide, Glimepiride, Glipizide, Glyburide, Tolbutamide and Tolcyclamide; (d) amino acid and benzoic acid derivatives, such as Nateglinide and Repaglinide; (e) α-glucosidase inhibitors, such as Acarbose; (f) cholesterol lowering agents, such as (i) HMG-CoA reductase inhibitors, e.g., Lovastatin, and other statins), (ii) bile acid sequestrants, e.g., Cholestyramine (iii) nicotinic acid, (iv) proliferator-activator receptor α-agonists, such as Benzafibrate, and Gemfibrozil, (v) cholesterol absorption inhibitors, e.g., β-sitosterol and (vi) acyl CoenzymeA:cholesterol acyltransferase inhibitors, e.g., Melinamide; and (vi) Probucol.
Whilst continuous efforts are directed at developing new anti-diabetic agents, there is also a considerable need for the development of materials related to known therapeutic agents that may display improved bioavailability, functionality or reduced levels of undesirable effects. There is also a need for new diagnostic agents that can facilitate elucidation of the mechanism of insulin release or sensitization and the binding mechanism of the known anti-diabetic agents to their respective molecular receptors, such as that of the sulfonylureas to the sulfonylurea receptor (SUR). The fact that hypoglycemic SUs exhibit substantially higher selectivity for KATP channels in pancreatic β-cells compared to other tissue makes them ideal candidates for specific β-cell probes.
Sulfonylureas have been labeled with radioisotopes (e.g., [3H]Glibenclamide is available from DuPont/NEN, Boston, Mass.), and modified to facilitate chromatographic or mass spectral analyses, using conventional derivatization methods, see e.g., Braselton et al. (Braselton W. E. Jr, Bransome E. D. Jr, Ashline H. C., Stewart J. T., Honigberg I. L., Gas chromatographic and mass spectral properties of sulfonylurea N-methyl-N′-perfluoroacyl derivatives, Anal. Chem., 48(9):1386-94, 1976). However, no sulfonylureas have been reported that contain fluorinated residues, e.g., 19F probes.
Fluorocarbon compounds and their formulations have numerous applications in medicine as therapeutic and diagnostic agents and as blood substitutes. Fluorine features a van der Waals radius (1.2A) similar to hydrogen (1.35A). Hydrogen replacement with fluorine does therefore not cause significant conformational changes; fluorination can lead to increased lipophilicity, enhancing the bioavailability of many drugs; and fluorinated materials are often biologically inert and are generally expected to reduce side-effect profiles of drugs. The carbon-fluorine bond strength (460 kJ/mol in CH3F) exceeds that of equivalent C—H bonds. Perfluorocarbons (PFCs) display high chemical and biological inertness and a capacity to dissolve considerable amounts of gases, particularly oxygen, carbon dioxide and air per unit volume. PFCs can dissolve about a 50% volume of oxygen at 37° C. under a pure oxygen atmosphere. Fluorocarbon compositions can be used for wound treatment, as described in U.S. Pat. No. 4,366,169. Fluorocarbon formulations are also useful in diagnostic procedures, for example as contrast agents (Riess, J. G., Hemocompatible Materials and Devices: Prospectives Towards the 21st Century, Technomics Publ. Co, Lancaster, Pa. USA, Chap 14 (1991); Vox Sanguinis, 61:225-239,1991).
Nuclear magnetic resonance (NMR) techniques permit the assessment of biochemical, functional, and physiological information from patients. Magnetic resonance imaging (MRI) of tissue water can be used to measure perfusion and diffusion with submillimeter resolution. Magnetic resonance spectroscopy may be applied to the assessment of tissue metabolites that contain protons, phosphorus, fluorine, or other nuclei. The combination of imaging and spectroscopy technologies has lead to spectroscopic imaging techniques that are capable of mapping proton metabolites at resolutions as small as 0.25 cm3 (Zakian K L; Koutcher J A; Ballon D; Hricak H; Ling C C, Semin Radiat Oncol.; 11(1):3-15, 2001). Molecular MR imaging employs contrast agents bound to targeting molecules that ‘light up’ specific cell types or sub-organ structures. Molecular imaging has been successfully used to monitor angiogenesis and inflammation based on unique surface molecules expressed in growing vascular tissue and in cells of the immune system (M. Singh, V. Waluch, Adv. Drug Del. Reviews, 41, 7-20, 2000.). In magnetic resonance angiography (MRA) contrast agents are used to image the arteries and veins for diagnosing cardiovascular disease and associated disorders.
PFCs are of considerable interest for 19F-MRI studies due to the attractive features of 19F as an in vivo MR probe. 19F's sensitivity is very high and rivals that of protons (83%) and little or no background 19F MR signal arises from fluorine of biological origin. Thus, 19F MR images arise only from exogenously administered PFCs, which offers a clear advantage over 1H-MRI techniques, as water, and hence 1H background signal, is abundant in biological tissue. Furthermore, since the observed signal intensity directly correlates with 19F spin densities, 19F MRI permits quantitation of the administered 19F probe. Of particular interest is fluorine's diagnostic value in non-invasive imaging applications. Apolar oxygen imparts paramagnetic relaxation effects on 19F nuclei associated with spin-lattice relaxation rates (R1) and chemical shifts. This effect is proportional to the partial pressure of O2 (pO2). 19F NMR can therefore probe the oxygen environment of specific fluorinated species in cells and other biological structures.
Nöth et al. (Nöth U; Grohn P; Jork A; Zimmermann U; Haase A; Lutz, J., 19F-MRI in vivo determination of the partial oxygen pressure in perfluorocarbon-loaded alginate capsules implanted into the peritoneal cavity and different tissues, Magn. Reson. Med. 42(6):1039-47, 1999) employed perfluorocarbon-loaded alginate capsules in MRI experiments to assess the viability and metabolic activity of the encapsulated materials. Quantitative 19F-MRI was performed on perfluorocarbon-loaded alginate capsules implanted into rats, in order to determine in vivo the pO2 inside the capsules at these implantation sites. Fraker et al. reported recently a related method with perfluorotributylamine (C. Fraker, L. Invaeradi, M. Mares-Guia, C. Ricordi, PCT WO 00/40252, 2000).
Although a large range of fluorinated products is available commercially, most PFCs suffer from a number of shortcomings. Many commercial PFCs currently employed for, diagnostic purposes were originally selected for blood substitution. Their physicochemical properties [J. G. Reiss et al., Biomat. Artif. Cells Artif. Organs, 16, 421-430, 1988.] are therefore not targeted towards specific diagnostic or other biomedical uses, particularly for MRI. The molecular features of these PFCs are not optimized for high-sensitivity 19F-MRI studies. Their T1 relaxation times are relatively long, T2 relaxation times are short, and severe J-modulation effects and chemical shift artifacts can profoundly limit their MRI utility. Whilst their immiscibility in water offers benefits in some respects, it necessitates the use of emulsifiers. Thus, for PFC-in-water emulsions, such as 1,2-bis-(perfluorobutyl)ethane (F-44E™), perfluorohexyl bromide, perfluorooctyl bromide (Perflubron™), perfluoromethyldecalin (PMD), perfluorooctyl ethane, perfluorotripropylamine, and the blood substitutes Fluosol™ and Oxygent™, lecithins or poloxamers are employed to disperse the PFCs and stabilize the emulsion. However, surfactants are problematic in that their use adds processing requirements and some of them can be unstable, chemically ill-defined or polydisperse, or cause potential undesirable side effects. The use of emulsions poses the additional disadvantage that the PFCs' fluorine content is effectively diluted (often by 50% or more), diminishing their spectral and imaging signal intensities and, hence diagnostic benefit. The impact of such dilutions is particularly evident in tumor oxygenation studies where only ˜10% of the injected PFC emulsion dose reaches the tumor, necessitating time consuming T1 measurements. This dilution effect is even more pronounced, when only a portion of the available PFCs' fluorine resonances is of diagnostic value. This is often the case, as severe chemical shift artifacts need to be circumvented by selectively exciting only a narrow chemical shift range containing one resonance (or a closely spaced group of resonances). Although F-44E™, for instance, has a high fluorine content (74%) with largely acceptable spectral features, many MRI studies. have selectively excited its trifluoromethyl resonance, representing only one third of the total F-content, which on emulsification (at 90%) is further diluted to ˜22%. Similarly, for MRI with perfluorononane the choice is between the selective acquisition of the single trifluoromethyl resonance (6 fluorines with a spectral width of 50 kHz at 7 Tesla) or multiple difluoromethylene resonances (14 fluorines with a 1300 kHz spectral dispersion) (see, e.g., S. L. Fossheim; K. A. Il'yasov, J. Hennig, A. Bjornerud, Acad. Radiol., 7(12):1107-15, 2000.).
Ideally, PFC imaging agents should combine the following features: non-toxic, biocompatible, chemically pure and stable, low vapor pressure, high fluorine content, reasonable cost and commercial availability. Additionally, they should meet several 19F-NMR criteria, including a maximum number of chemically equivalent fluorines resonating at one or only few frequencies, preferably from trifluoromethyl functions. Some of the other spectral criteria have been discussed in detail elsewhere (C. H. Sotak, P. S. Hees, H. N. Huang, M. H. Hung, C. G. Krespan, S. Raynolds, Magn. Reson. Med., 29, 188-195, 1993.). For MRI, it would furthermore be desirable to have control over the amount of magnetically responsive material for specific uses, and to employ temperature-responsive and pH-dependent imaging agents for special uses. These could have applications in MRI-based temperature monitoring for use in general hyperthermia treatment (see, e.g., S. L. Fossheim; K. A. Il'yasov, J. Hennig, A. Bjornerud, Acad. Radiol., 7(12),1107-15, 2000.) of tumors and for monitoring the efficacy of chemotherapy, respectively (see, e.g., N. Rhagunand, R. Martinez-Zagulan, S. H. Wright, R. J. Gilles, Biochem. Pharmacol., 57, 1047-1058, 1999; I. F Tannock, D. Rotin, Cancer Res., 49, 4373-4383, 1989.). Furthermore, water solubility would enhance the PFC functionality in many biomedical settings, as it would obviate the need for emulsifiers.
Although selected efforts have been directed at developing new fluorinated MRI probes, none are water soluble compounds [e.g., perfluoro-[15]-crown-5 ether)], and some are commercially unavailable [e.g., perfluoro-2,2,2′,2′-tetramethyl-4,4′-bis(1,3-dioxalane)]. It appears no attempts have so far focused on screening available PFCs from the thousands of commercial fluorinated products in order to identify potentially more suitable MRI probes for biomedical uses. It seems furthermore that no studies have attempted to establish structure activity relations (SARs) of related PFCs for MRI purposes. Noteworthy is also the fact that all PFCs examined to date have molecular weights under 1,000, typically between 400-600 Da. This is partly a reflection of the specific requirements for blood substitution agents, but also due to the widely held belief that higher molecular weight or polymeric fluorinated agents would not be detectable by 19F-NMR due to anticipated excessive line broadening, and would therefore be unsuitable. Thus, with the exception of the polymer-encapsulated PFCs noted above, this important class of materials had so far been excluded from consideration.
Paramagnetic ions, such as gadolinium (Gd3+) decrease the T1 of water protons in their vicinity, thereby providing enhanced contrast. Gadolinium's long electron relaxation time and high magnetic moment make it a highly efficient T1 perturbant. Since uncomplexed gadolinium is very toxic, gadolinium chelate probes, such as gadolinium diethylenetriamine pentaacetic acid (GdDTPA Mw 570 Da), albumin-GdDTPA (Gadomer-17, Mw 35 or 65 kDa), have been employed extensively in MRI of tumors and other diseased organs and tissues. Several other developmental chelators have also been reported, including dual-labeled agents, oligonucleotide-derived, dextran-derived GdDTPA, and TAT and other peptide-derived chelators. However, presently approved MRI contrast agents are either not tissue specific, e.g., GdDTPA, or target only normal tissue, which limits their utility in diagnosis of metastases or neoplasia. MRI studies with GdDTPA, for instance, do not correlate with the angiogenic factor or the vascular endothelial growth factor (VEGF). Attempts have also been made to overcome the low relaxivities of small Gd-DTPA chelates by preparing polymer conjugates of Gd(DTPA)(2−) [see e.g., MRA. Duarte M. G.; Gil M. H.; Peters J. A.; Colet J. M.; Elst L. Vander; Muller R. N.; Geraldes C. F. G. C., Bioconjug. Chem., 21, 170-177, 2001.]. However, the relaxivity of these polymer conjugates was only slightly improved and they were also cleared very quickly from the blood of rats, indicating that they are of limited value as blood pool contrast agents for MRI. The clinical use of polymer-coated paramagnetic iron oxide particles as a tissue-specific MRI contrast agent is well established (R. Weissleder, et al., Radiology, 175, 494-498, 1990.). MRI with iron-oxide particles has been successfully used to image apoptic cells (M. Zhao et al., Nature Medicine, 7, 1241-1244, 2001.) and rat T-cells at the cellular level (S. J. Dodd et al., Biophysical J., 76, 103-109, 1999.). Although the need has been recognized, non-invasive MRI techniques have so far not been applied to β-cells and islets.
Whilst much can be achieved with currently available imaging and contrast agents, there are still unmet needs for novel diagnostic agents, particularly for those exploiting biological specificity. Imaging agents suitable for targeting receptors involved in insulin production and utilization would substantially enhance our understanding of the diabetes disease process and the function of anti-diabetic drugs. The development of imaging techniques and diagnostic reagents for non-invasive in vivo assessment of β-cell mass may be instrumental in managing pancreas and islet transplantation, in the understanding of the pathogenesis in islet engraftment, and for assessing the efficacy of modulations in type 1 diabetes therapy. Although selected efforts have been directed at developing such new probes, a broader investigation of these agents is urgently needed.